Insulin plays a central role in the treatment of diabetes mellitus (DM). If feasible at the molecular level, ultra-stable insulin analogs could (a) enhance the safety of intraperitoneal pumps for the treatment of Type 1 DM and (b) facilitate global distribution of insulin formulations for the treatment of Type 1 and Type 2 DM. This proposal, in a nutshell, seeks to translate molecular insights to obtain ultra-stable therapeutic insulin analogs. Our approach focuses on the structure and function of ultra-stable single-chain insulin (SCI) analogs. SCIs, containing a shortened connecting peptide (?C domain?) between A- and B chains, also provide unique probes of structure-activity relationships. Protein design rests upon two premises: Hypothesis 1: That the SCI framework provides a ?sweet spot? for design of C domains: long enough to enable induced fit on receptor binding yet incompatible with amyloid-associated thermal inactivation. Hypothesis 2: That SCIs can be made ultra-stable via sequence features of the A, B and C domains to damp conformational fluctuations in the free protein and yet permit native biological activity. The proposed studies will provide design rules for clinical translation. Our preliminary results suggest that SCIs can provide a platform for all 3 therapeutic forms of insulin: rapid-acting (prandial), long-acting (basal) and biphasic (pre-mixed) formulations. Attention will be paid to cell-specific signaling properties, including potential mitogenicity relative to WT human insulin and carcinogenic analog X10 (AspB10-insulin). The essential idea underlying our SCI strategy exploits a conformational switch between a closed state of the hormone (as observed in classical structures of insulin) and an open state (as observed on binding of the hormone to a model IR fragment complex; designated the ?micro-receptor? (IR)). Our strategy and its feasibility are described in two recent back-to-back publications: Glidden, M.D., et al. J. Biol. Chem. 293, 47-68 and 69-88 (2018ab). Methods are described in detail in these articles and their web-based Supplements. Protein design will be based both on classical insulin crystal structures (Adams, M.J., et al. Nature (1969) 224, 491-5) and on recent advances in the structural dissection of the insulin receptor (IR) and its mode of hormone binding [Menting, J.G., et al. Nature (2013) 493, 241-5, and PNAS-Plus (2014) 111, E3395-404], with innovation enhanced via cryo-EM structures of the intact ectodomain containing insulin bound in its signaling conformation [Weis, F., et al. Nature Commun. (2018) 9, 4420]. The Research plan thus exploits the ongoing cryo-EM ?resolution revolution? via Subcontract to M.C. Lawrence. An interdisciplinary Approach is proposed by a unique team with integrated MPI Management Plan and Internal Advisory Committee. The significance and innovation are described by American Diabetes Association President Dr. L. Philipson (University of Chicago; letter attached).